In recently years, full length monoclonal antibodies have been successfully used to treat cancer, autoimmune and inflammatory diseases and other human diseases. Although there are five different types of immunoglobulins (IgA, IgD, IgG, IgM and IgE) existed in nature, IgG represents the most suitable modality for human therapeutics because of the favorable properties concerning the high binding affinity and specificity, high bioavailability, long serum half life in circulation, potential effector function capability and the industrial-scale manufacturability.
Conventional IgGs are tetramer molecules comprising two identical heavy chains and two identical light chains. IgG heavy chain has a variable domain at the N-terminus followed by the first constant domain (CH1), a hinge and two additional constant domains (CH2CH3). IgG light chain is composed of two domains: an N-terminal variable domain and a C-terminal constant domain. The heavy chain variable region (VH) interacts with the light chain variable region (VL) and the first constant region of heavy chain (CH1) interacts with the light constant (CL) to form the Fab structure. Two CH2CH3 domains form homodimeric Fc structure. So an IgG has two antigen binding Fab arms that are relatively flexible in orientation with each other and with the Fc domain. This structural feature renders IgG the capability of activating certain types of receptors on cell surface due to target dimerization induced by bivalent binding (when both Fab arms bind to antigen). Furthermore, because IgG Fc can bind to cell surface Fc gamma receptors (FcγR), cell surface antigens bound to IgG can be cross-linked to form receptor cluster and thus activated. Conceptually, FcγR can also mediate receptor cross-linking in the event of monovalent antibody/antigen binding. Activation of cell surface receptors by IgG (agonistic antibody) has been successfully demonstrated on a large number of cell surface targets (Seo, Nat. Med. (2004) 10:1088-94, TRAIL receptor (Belyanskay, Mol. Cancer (2007) 6:66-78, Manero, Cell Stress Chaperones (2004) 9:150-66, Westwood, Journal of translational Med. (2010) 8:42-9, Budach, J. Radiat. Oncol. Biol. Physc (2009) 75:198-2002).
While receptor agonist is a useful property of IgG, it is undesirable for some applications. For example, anti-cMet antibody intended to block HGF/cMet signaling in cancer cells actually leads to the activation of this signaling pathway (Martens, Clin. Cancer Res. (2006) 12:6144-52). Anti-TNFR-1 antibodies intended for selectively blocking the TNF ligand signaling through this receptor while sparing TNFR-2 signaling, which is believed to be beneficial to suppressing inflammation, can induce target receptor signaling (Kontermann, J. Immunother. (2008) 31:225-34; Faustman, Nat. Rev. drug discovery (2010) 9:482-493).
A conventional wisdom to avoid the unwanted target cross-linking is to use engineered antibody structures where only one binding unit exists for a given specificity. These antibody structures are commonly referred to as monovalent antibodies. Monovalent antibodies offer significant expansion of the toolbox for treating human diseases.
A straightforward and efficient engineering strategy for monovalent antibodies is to use single variable domain, single chain Fv (scFv) or Fab fragment. An anti-TNFR-1 domain antibody and Fab fragments were engineered to selectively block TNF signaling through TNFR-1 (sparing the signaling through TNFR-2) (Kontermann, J. Immunother. (2008)31(3):225-34; US20080008713; WO2008149144; US20100150916). The major drawback for these antibody fragments for systemic therapeutic application is the short half life because of their small size (below kidney filtration threshold of ˜60 kD). To make these antibody fragments practically useful therapeutics for chronic diseases, half-life extension strategies are needed (Kontermann, BioDrugs (2009) 23:93-109). The strategies include pegylation (US24121415), fusion with albumin (Muller, J. Biol. Chem. (2007) 282:12650-60) or fusion with albumin binder (Stork, Prot. Eng. Des. Sel. (2007) 20: 569-76).
Fusion with monomeric Fc (CH3 interface engineered to disrupt CH3-CH3 association, US 2009/022642) or single chain Fc (scFc) (in sequence configuration of N-terminus-hinge-CH2-CH3-linker-hinge-CH2-CH3-C-terminus provides a potential solution to generating monovalent antibodies) is another potential solution for generating monovalent antibodies with improved half life (WO2005077981, WO/2008/012543, US20090304696, US20090252729).
Additional engineering approaches focusing on the CH3-CH3 interface residues have been undertaken to make monovalent antibodies. CH3 interface engineering creating a “knob” in one and a “hole” in the other CH3 led to the formation of heterodimeric Fc molecule (knob-in-hole, U.S. Pat. No. 5,821,333, Merchant et al., Nat Biotech. (1998)16:677-681). Asymmetrical fusion of antibody V domain-containing fragments to the mutant CH2CH3 chain can lead to various monovalent antibody molecules. Based on this technology, one-arm antibody, OA5D5, specific to cMet has been generated (U.S. Pat. No. 5,821,333, US2008/0063461). Rigorous preclinical tests strongly suggest that, unlike the bivalent antibody counterpart, this monovalent antibody is a pure cMet antagonist (without agonistic activity). This monovalent antibody is currently being tested in multiple anti cancer clinical trials. A similar heterodimeric Fc engineering strategy based on amino acid substitutions in CH3-CH3 interface was used to create an anti-TNFR-1 monovalent antibody (WO2008089004, Gunasekaran et al., J.B.C. (2010) 285:19637-19646). Efficient Fc heterodimer formation was also devised based on strand-exchange engineered domain (SEED) design (Davis et al., PEDS 2010, 23:195-202, US20070287170).
It is noted that the success of the above mentioned monovalent antibodies reflects the real demand, significant investment effort and therefore scientific advancement. However, generation of these molecules involves either chemical modification or amino acid substitutions at conserved positions which often decrease protein stability. Destabilized proteins as therapeutics may raise concerns over product manufacturability and clinical safety (such as immunogenicity). Therefore, there remains a need for new antibody modalities which will not activate target receptors upon binding and in the meantime offer improved profiles on product stability, safety and manufacturability.